Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly

Wayne A. Duffus, Pnina Levy-Mintz, Matthew R. Klimjack, Margaret Kielian

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37°C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28°C) dramatically decreased El cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28°C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28°C. The altered- assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.

Original languageEnglish (US)
Pages (from-to)2471-2479
Number of pages9
JournalJournal of virology
Volume69
Issue number4
DOIs
StatePublished - Apr 1995

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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